Intravital imaging of cell-specific metabolic activity is of key importance for understanding of a wide range of clinical conditions. Among them is compromised blood perfusion following a stroke and a decrease in efficiency of single-cell respiratory processes that occurs in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. However, no methods are available today for in vivo measurement of single cell metabolism with sufficient sensitivity to resolve fast metabolic events related to ongoing neuronal electrical activity and neuronal responses to stimulation. To meet this challenge, we will adapt an existing technology, 2- photon laser scanning microscopy, for high-resolution microscopic imaging of functional metabolism of single brain cells, taking advantage of intrinsic fluorescence of metabolic cofactor 2-nicotinamide adenine dinucleotide (NADH). This project combines a bioengineering effort with testing of biological hypotheses and brings together an interdisciplinary team of experts in neuroscience, physics, engineering and computational science. From the engineering perspective, we propose to (1) develop and validate 2-photon imaging of NADH with sufficient sensitivity to detect functional changes from single cortical neurons and astrocytes in response to sensory stimulation in living animals, and (2) optimize the optical design and experimental protocol for simultaneous in vivo 2-photon imaging of metabolic, neuronal and vascular activity with high spatial and temporal resolution. Using these technological developments, we will explore new concepts concerning the mechanisms of neuro-vascular-metabolic coupling. Specifically, we will (1) address the relative contribution of oxidative phosphorylation and glycolysis to the transient metabolic response in neurons and astrocytes, (2) test the relationship between astrocytic metabolic response and regulation of blood flow through astrocytic calcium-dependent mechanisms, and (3) establish functional NADH imaging as a biomarker for hypoxia and mitochondrial dysfunction. These goals will be achieved by focusing on temporal signal characteristics, by investigation of simultaneously acquired signals, and by using in vivo pharmacology. The main deliverable of the proposed project - a tool for simultaneous 2-photon imaging of metabolic, neuronal and vascular activity - has a potential to transform the investigation of rodent models of human brain disease by opening an unprecedented opportunity to study the homeostasis and functional interactions among neurons, glia, and capillaries of the living brain. In future, this tripartite imaging approach repeated at different stages of disease would allow establishing a defined set of in vivo imaging biomarkers characterizing the progression of neuro-vascular-metabolic pathology that could be used for objective screening of potential therapies. PUBLIC HEALTH RELEVANCE: We will adapt an existing technology, 2-photon laser scanning microscopy, to visualize functional metabolism of single brain cells in living animals taking advantage of the intrinsic fluorescence of metabolic cofactor 2- nicotinamide adenine dinucleotide (NADH). Furthermore, we will combine NADH imaging with 2-photon measurements of neuronal and vascular activity and will establish NADH as a microscopic imaging biomarker for hypoxia or a decrease in mitochondrial efficiency in a mouse model of human disease.